Recommended
Recommended
More Related Content
What's hot
What's hot (20)
Similar to Toward Precision Medicine in Neurological Disease by David Goldstein
Similar to Toward Precision Medicine in Neurological Disease by David Goldstein (20)
Recently uploaded
Recently uploaded (20)
Toward Precision Medicine in Neurological Disease by David Goldstein
- 1. Towards precision medicine in neurodevelopmental disease David B. Goldstein Duke University (January Columbia University)
- 2. Questions! If you have any questions during the webinar, please enter them in the GoToWebinar pane. We will answer as many as possible at the end.
- 3. Project 1: Epilep8c Encephalopathies Erin Heinzen Elizabeth Ruzzo Andrew Allen Slave Petrovski Yujun Han Jonathan Keebler Yi-‐Fan Lu Epi4K Consor,um. Epi4K: gene discovery in 4,000 genomes, Epilepsia, 2012 Aug;53(8):1457-‐67.
- 4. Gene8cs of Epilep8c Encephalopathies Performed Sequence Analysis on 356 trios with one of two classic encephalopathies • Infan8le Spasms (IS): CharacterisQc chaoQc interictal & EEG paSern of hypsarrhythmia, the sine qua non of the syndrome • Lennox-‐Gastaut syndrome (LGS): Characterized by mixed seizure types and intellectual disabiliQes Epi4K Consor,um. De novo muta,ons in epilep,c encephalopathies. Nature. 2013 Sep 12;501(7466): 217-‐21 EuroEpinomics and Epi4K, accepted for publica,on in AJHG
- 5. Distribu8on of de novo muta8ons in 356 trios EuroEpinomics and Epi4K, accepted for publica,on in AJHG
- 6. Effec8ve Gene Muta8on Rate EuroEpinomics and Epi4K, accepted for publica,on in AJHG
- 7. Rare Muta8ons, More Common Pathways 12% of paQents geneQcally explained by a causal de novo mutaQon Causal mutaQons very rare, but clearly organized into specific biological process including for infanQle spasms and LGS – 8 / 356 paQents with GABA receptor mutaQons – > 12 / 356 with mutaQons influencing vesicle trafficking (or membrane dynamics more generally)
- 8. Muta8ons Influencing Vesicle Trafficking DNM1 STXBP1 * = SNV * = stop gained
- 9. DNM1 Func8on • ParQcipates in receptor-‐mediated endocytosis. Localizes to pre-‐synapQc terminal • Mechanochemical protein – binding of GTP causes a conformaQonal change, allowing DNM1 to pinch vesicles from membrane • Fihul mouse mutaQon causes epilepsy in mice (Boumil, 2001) DNM1
- 10. DNM1 Muta8ons 314 499 631 746 864 G domain" Middle domain" PH" GED" PRD" A177P" G359A" K206N"
- 11. Quan8fica8on via High-‐Content Imaging 110 100 90 80 70 60 50 40 30 20 10 0 Wildtype A177P K206N G359A % WT Intensity Transferrin Uptake 120 100 80 60 40 20 0 WT A177P K206N G359A % WT Punctae Per Cell
- 12. • All Implica8ons three mutaQons show possible dominant negaQve effects on endocytosis. • Vesicle trafficking dysfuncQon as a mechanism of epilepsy (DNM1, STXBP1, SYN1). • Mouse Qfl mutaQon shows aberrant vesicle formaQon in brain (Boumil, unpublished data).
- 13. Iden8fying “signatures” of causal muta8ons amongst de novo muta8ons PrioriQzing mutaQons using both intolerance score (gene level) and Polyphen (variant level) Petrovski et al. Genic intolerance to func,onal varia,on and the interpreta,on of personal genomes. PLOS Gene,cs 2013;9(8):e1003709
- 14. Degree to which genes have more, or less, common func8onal varia8on than expected given the amount of presumably neutral varia8on they carry
- 15. Among the control populaQon 1.9% of sequenced controls had a hot zone de novo mutaQon occurring in an essenQal gene.
- 16. Among the control populaQon 1.9% of sequenced controls had a hot zone de novo mutaQon occurring in an essenQal gene. This is compared to the 15.2% of cases (Fisher’s Exact, p = 4.5x10-‐8; 88% excess observa8ons among cases).
- 17. First iden8fied deglycosyla8on syndrome • Symptoms: PaQent had compound heterozygous variants in N-‐glycanase 1 (NGLY1). He inherited a frameshiq variant from his mother, and a nonsense mutaQon from his father Developmental delays, involuntary movements, mulQfocal seizures, abnormal liver funcQon, absent tears. • Previously tested negaQve for congenital disorders of glycosylaQon Need et al. Clinical applica,on of exome sequencing in undiagnosed gene,c condi,ons J Med Genet. 2012 Jun;49(6):353-‐61
- 20. KCNT1 and Epilep8c Encephalopathies • MutaQons in KCNT1 have been implicated in two epilepsy disorders – Autosomal Dominant Nocturnal Frontal Lobe Epilepsy (ADNFLE) – Epilepsy of Infancy with MigraQng Focal Seizures (EIMFS) • Expression of mutant and wild type protein in oocytes shows that all idenQfied mutaQons are Gain of FuncQon (Petrou Lab)
- 21. KCNT1 and Quinidine • KCNT1 GoF mutaQons are quinidine sensiQve Milligan et al. KCNT1 gain of func,on in 2 epilepsy phenotypes is reversed by quinidine. Annals of Neurology. 2014 March 3 epub ahead of print
- 22. KCNT1 Pa8ent 1 • Intractable seizures, global developmental delay, cerebral atrophy, severe microcephaly • de novo mutaQon in KCNT1 (K629N, c.1887 G>C) KCNT1 Pa8ent 2 • Over 40 nocturnal seizures a night by age 4, regressed to non-‐ ambulatory non-‐verbal state • De novo mutaQon in KCNT1 was found ( Y796H, c.2386 T>C)
- 23. KCNT1 Y796H Courtesy of Steve Petrou, University of Melbourne
- 24. Response to Quinidine Therapy • Quinidine 12 mg/kg/d on first day • Quinidine 19 mg/kg/d on second day • Seizures dropped to 2/d, level 1.6 ug/ml (cardiac therapeuQc 2-‐5). • Dose maintained on 12 mg/kg/day • Seizures over the next 2 weeks: 3/day, level 0.4 ug/ml • Improved alertness head control siung and interacQon observed • Plan to increase dose while monitoring ECG • Duke a KCNT1 Related Epilepsy Clinic for other paQents started 7 6 5 4 3 2 1 0 Seizures/day
- 25. 0.2 0.15 0.1 0.05 0 0.05 0.1 Q543R chr9:g.138662152A>G -‐0.1 -‐0.08 -‐0.06 -‐0.04 -‐0.02 0 0.02 0.04 Caucasian compared to *Middle Eastern* Ashkenazi (selfDec) KCNT1_Q543R_posi8ve
- 26. SeqClinic – “rapid mode” sample • 18 month old girl with sudden onset of opsoclonus, ataxia, weakness of the upper extremiQes that have been waxing and waning. • Highly progressive • Consider for aggressive treatment on presumpQon of an autoimmune condiQon • Enrolled in sequencing protocol on March 27 and flagged as “urgent” • compound heterozygote for LoF mutaQons in SLC52A2 – Gene has 9.5%Qle RVIS – Missense reported pathogenic in four of 14 unrelated cases recently summarized. – Stop gain variant not previously implicated..
- 27. SLC52A2 is required for Riboflavin uptake • Compound het genotypes have been shown to produce essenQally no SLC52A2 protein • FuncQonal assessment in previous literature illustrated that observed missense variant “abolished” riboflavin uptake. • Early death from respiratory failure may occur • Some pa8ents show significant clinical improvement with riboflavin supplementa8on
- 28. TIMELINE Day 1 – Bloods drawn from family Day 3 – Samples submiSed for sequencing Day 7 – Samples fragmented – sequencing iniQated on trio Day 12 – Released to BioinformaQcs team Day 15 – Available for analysis Day 20 – Analyses completed Day 22 – Compound het in disease gene reported to Clinical team Day 24 Family visit clinic – Riboflavin treatment ini8ated
- 29. Riboflavin Treatment A B C D A. 3 weeks pre-‐treatment. B. Day 2 of treatment. C. 3 weeks post-‐treatment D. 4 weeks post-‐treatment
- 30. EGI Repository • Current clinical sequencing only looks for geneQc changes known to be solidly associated with epilepsy, leaving much DNA unexplored • The EGI will create a data repository of clinical exome and genome sequences • Data will be reanalyzed every 6 months for novel geneQc changes • New results will be communicated back to paQents via their doctor. • Data will also be made available to advance epilepsy research
- 31. Efforts by: • David EGI Par8cipa8ng Sites Goldstein (Duke) • Erin Heinzen (Duke) • Dan Lowenstein (UCSF) • Sam Berkovic (University of Melbourne) • Tracy Dixon-‐Salazar (CURE) Inaugural Academic Medical Center Partners: • Duke University (Bill GallenQne and Mohamad MikaQ) • University of San Francisco (Joe Sullivan) • Children’s Hospital of Philadelphia (Dennis Dlugos) • Children’s Hospital, Boston (Anna Poduri) • New York University (Orrin Devinsky) • University of Melbourne (Ingrid Scheffer) IRB approved at Duke University First sequence making its way though analysis.
- 32. • Establish standards for geneQc diagnosQcs • Increase the speed of geneQc diagnoses • Establish generalizable modeling frameworks • Establish mechanism to organize clinical experiences of geneQcally informed therapies
- 33. Mul8 Electrode Array (MEA)
- 34. Celf4 mouse model and seizure • Celf4: – Encode an RNA-‐binding protein – Involves in RNA processing, such as pre-‐mRNA splicing, RNA stability, and translaQon – widely expressed in the brain • Celf4 deficiency and seizures in mice was described Dr. Wayne Frankel’s lab – A new epilepsy mouse model called ‘‘frequent-‐flyer’’ – DisrupQon of the coding region of Brunol4 (Celf4) – Limbic and tonic–clonic seizure started from the third month – Younger mice had lowered seizure threshold
- 35. Elevated number of spikes in network spike • Four independent MEA experiments showed elevated spikes in network spike in Celf4 knockout neurons compared to wild-‐type Days in vitro (DIV) Days in vitro (DIV) Mean spikes in network spike Mean spikes in network spike Mean spikes in network spike 2014/07/16 2014/07/30 2014/07/28 2014/08/26
- 36. RaQonale to test FluoxeQne (Prozac) on MEA • CELF4 deficiency down-‐regulates serotonin receptor 2c • FluoxeQne % protein expression to wt (Prozac) blocks serotonin reuptake • FluoxeQne (Prozac) can reduce seizure phenotype in mice (measured by spike-‐wave discharges/hour) by 50%
- 37. FluoxeQne prevented network phenotype to occur in Celf4 KO neurons • Treat FluoxeQne (3 uM) in Celf4 neurons on MEA at DIV15
- 38. IdenQfied human CEFL4 mutaQons in epilepsy paQents • Idiopathic generalized epilepsy 18_34844669_A het epprnd29001uw1 case missense • Nonlesional focal epilepsy 18_34846495_T het epprnd35229awb1 case splice donor Family history + 18_34846517_T het epprnd41146bgn1 case missense Family history + 18_34854293_C het dukeepi119 case missense No Family history 18_35145382_T het epprnd36043ary1 case missense Family history + One pa,ent with a CELF4 missense muta,on is seizure-‐free during 10 months of Prozac treatment
- 39. Acknowledgements Alterna8ng Hemiplegia Erin Heinzen Sophie Nicole Nicole Walley Fiorella Gurrieri Mohamad MikaQ Mary King Sanjay Sisodiya David Webb Kathryn Swoboda Ingrid Scheffer Samuel Berkovic Peter Uldall Boukje de Vries Domininque Ponceli Arn M.J.M. van den Maagdenbergn Genomic Analysis Facility Erin Heinzen Brian Krueger Joshua Bridgers Linda Hong Erin Campbell Evan Kazura Romelia Perez-‐Marco Bioinforma8cs Team Liz Cirulli Mingfu Zhu Jessica Maia Jonathan Keebler Hee Shin Kim Zhong Ren Nanye Long Richard and Pat Johnson Duke, UCB Vandana Shashi Mohamad MikaQ Yong-‐Hui Jiang Rebecca Spillmann Kelley Schoch Slave Petrovski Epi4K NINDS, 5U01NS077303-‐02 5U01NS077274-‐02 Erin Heinzen, Elizabeth Ruzzo Slave Petrovski Andrew Allen Yujun Han Epi4K ConsorQum